PROJECT SUMMARY/ABSTRACT Post-translational modification by ubiquitin is an essential mechanism to alter protein function in eukaryotes. Ubiquitin, a 76 amino acid protein, is attached to specific proteins via a cascade of ubiquitin activating enzyme E1, conjugating enzyme E2, and ubiquitin ligase E3. Ubiquitination plays an essential role in a broad aspect of cellular processes, including transcription, DNA repair, signal transduction, autophagy, cell cycle, immune response, and membrane trafficking. Aberration in the ubiquitination system lead to a number of human diseases, such as neurodegenerative diseases and cancers. Remarkably, although ubiquitin is absent in prokaryotes, Bacterial pathogen encoded E3 ubiquitin Ligase (BELs) are found to be delivered into eukaryotic host cells to manipulate the host ubiquitin system for successful infection. We recently discovered a novel family of BELs, named SidC, from the intracellular bacterial pathogen Legionella pneumophila. SidC have no sequence and structure resemblance to any known eukaryotic RING-type or HECT-like ubiquitin ligases. Our preliminary data raised intriguing questions such as what is the unique molecular mechanism of this novel family of ubiquitin E3 ligases given their apparent disparities to canonic HECT or RING-finger E3 ligases at both primary and tertiary structure levels? What are the specific substrates of SidC? And how the ubiquitination of these host potential host factors play a role in membrane trafficking regulation? The overarching goal of this proposal is to elucidate the molecular mechanism of this distinct family of ubiquitin ligases and their role in exploiting host membrane tracking. Specifically, we will pursuing the following aims: Aim 1: To delineate the molecular mechanism of ubiquitin ligation by SidC/SdcA. Aim 2: To elucidate the activity regulation of SidC/SdcA. Aim 3: To characterize the role of SidC/SdcA in host membrane trafficking and to identify their specific substrates. We expect the successful implementation of this proposal will make significant contributions to the understanding of both the molecular mechanisms of the enzymatic cascade of ubiquitination and the role of host ubiquitin pathway in the pathogenesis of bacterial pathogens.